A normal blood vessel is composed of a tubule of endothelial cells in contact with a supporting layer of mural cells (pericytes or smooth muscle cells) that stabilize the vessel and render the vessel quiescent. In contrast, the vasculature formed by a tumor is highly disorganized and exhibits decreased and abnormal association with mural cells. This defect in tumor vasculature causes several challenges in tumor control: i) while in normal tissues, mural cells prevent the endothelium from proliferating, lack of functional mural cell association allows tumor to stimulate endothelial proliferation and generate new vessels to feed the tumors; ii) the lack of mural cells makes vessels more susceptible to tumor intravasation, leading to metastasis; and iii) the leakiness of defective tumor vessels decreases delivery of therapeutic drugs. We seek to understand therapeutically overcome these defects. Our preliminary data demonstrate that the junctional molecule Connexin 43 (Cx43) plays a previously unidentified role in stabilization of normal blood vessels. Moreover, breast and brain tumor cells downregulate or inactivate mural cell Cx43 to disrupt their interaction with endothelial cell. Based on these novel findings we will investigate the hypothesis that tumor-induced inhibition of Cx43 plays a major role in destabilization of tumor blood vessels and that restoration of vascular Cx43 will reverse the deleterious effects. To investigate this hypothesis, we propose three specific aims: 1) To identify regulators of Cx43 activity in tumor-exposed vascular cells; 2) To determine whether altered host Cx43 affects the growth, angiogenesis, or metastasis of syngeneic mammary tumors; and 3) To determine whether altered host Cx43 affects vessel integrity and permeability. In Aim 1 we will use pharmacological inhibitors, site-specific Cx43 phosphorylation mutants, and gap junction-deficient Cx43 mutants to define the mechanism(s) by which tumor alters Cx43 expression and function. We will also test the ability of compounds reported to upregulate Cx43 and/or enhance vessel stability to override tumor-induced loss of functional Cx43 in vascular cells. In Aims 2 and 3, we will use a syngeneic mouse mammary tumor model to address whether tumor angiogenesis, metastasis, vascular permeability and interstitial fluid pressure are enhanced on a Cx43 host-deficient background, and whether Cx43 overexpression will prevent or delay these events via vascular stabilization. Together, these studies will define the role of Cx43 in blood vessel stability and elucidate the mechanism by which its function is lost in a pathological vasculature. Success of the proposed studies will identify Cx43 as a novel target to normalize tumor vasculature, thereby inhibiting angiogenesis, and potentially decreasing metastasis and enhancing drug delivery, ultimately leading to better cancer control. PUBLIC HEALTH RELEVANCE: The leaky, unstable blood vessels found in tumors facilitate tumor growth, impair drug delivery, and facilitate metastasis. Success of the proposed studies will identify vascular Connexin 43 as a novel target to normalize the tumor vasculature and thereby inhibit angiogenesis, decrease metastasis, and enhance drug delivery, ultimately leading to better cancer control.